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- EMDB-44019: INF2 at the Barbed End of F-Actin with Incoming Actin -

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Basic information

Entry
Database: EMDB / ID: EMD-44019
TitleINF2 at the Barbed End of F-Actin with Incoming Actin
Map data
Sample
  • Complex: Actin
    • Complex: INF2 Dimer
      • Protein or peptide: Inverted formin-2
      • Protein or peptide: Inverted formin-2
    • Complex: Actin Filament
      • Protein or peptide: Actin, alpha skeletal muscle
  • Ligand: ADENOSINE-5'-DIPHOSPHATE
  • Ligand: MAGNESIUM ION
KeywordsActin / Filament / Elongation / Ends / CYTOSOLIC PROTEIN
Function / homology
Function and homology information


cytoskeletal motor activator activity / tropomyosin binding / myosin heavy chain binding / regulation of mitochondrial fission / troponin I binding / mesenchyme migration / filamentous actin / actin filament bundle / striated muscle thin filament / actin filament bundle assembly ...cytoskeletal motor activator activity / tropomyosin binding / myosin heavy chain binding / regulation of mitochondrial fission / troponin I binding / mesenchyme migration / filamentous actin / actin filament bundle / striated muscle thin filament / actin filament bundle assembly / skeletal muscle thin filament assembly / skeletal muscle myofibril / actin monomer binding / stress fiber / skeletal muscle fiber development / titin binding / actin filament polymerization / filopodium / actin filament / Hydrolases; Acting on acid anhydrides; Acting on acid anhydrides to facilitate cellular and subcellular movement / small GTPase binding / calcium-dependent protein binding / lamellipodium / actin binding / cell body / hydrolase activity / protein domain specific binding / calcium ion binding / positive regulation of gene expression / perinuclear region of cytoplasm / magnesium ion binding / ATP binding / identical protein binding / cytoplasm
Similarity search - Function
Formin, FH3 domain / Formin, GTPase-binding domain / Diaphanous FH3 Domain / Diaphanous GTPase-binding Domain / Diaphanous FH3 Domain / Diaphanous GTPase-binding Domain / Rho GTPase-binding/formin homology 3 (GBD/FH3) domain / Rho GTPase-binding/formin homology 3 (GBD/FH3) domain profile. / Formin, FH2 domain / Formin, FH2 domain superfamily ...Formin, FH3 domain / Formin, GTPase-binding domain / Diaphanous FH3 Domain / Diaphanous GTPase-binding Domain / Diaphanous FH3 Domain / Diaphanous GTPase-binding Domain / Rho GTPase-binding/formin homology 3 (GBD/FH3) domain / Rho GTPase-binding/formin homology 3 (GBD/FH3) domain profile. / Formin, FH2 domain / Formin, FH2 domain superfamily / Formin Homology 2 Domain / Formin homology-2 (FH2) domain profile. / Formin Homology 2 Domain / WH2 motif / WH2 domain / WH2 domain profile. / Actins signature 1. / Actin, conserved site / Actins signature 2. / Actin/actin-like conserved site / Actins and actin-related proteins signature. / Actin / Actin family / Actin / ATPase, nucleotide binding domain / Armadillo-like helical / Armadillo-type fold
Similarity search - Domain/homology
Actin, alpha skeletal muscle / Inverted formin-2
Similarity search - Component
Biological speciesHomo sapiens (human) / Oryctolagus cuniculus (rabbit)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.82 Å
AuthorsPalmer NJ / Barrie KR / Dominguez R
Funding support United States, 1 items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS) United States
CitationJournal: Nature / Year: 2024
Title: Mechanisms of actin filament severing and elongation by formins.
Authors: Nicholas J Palmer / Kyle R Barrie / Roberto Dominguez /
Abstract: Humans express 15 formins that play crucial roles in actin-based processes, including cytokinesis, cell motility and mechanotransduction. However, the lack of structures bound to the actin filament ...Humans express 15 formins that play crucial roles in actin-based processes, including cytokinesis, cell motility and mechanotransduction. However, the lack of structures bound to the actin filament (F-actin) has been a major impediment to understanding formin function. Whereas formins are known for their ability to nucleate and elongate F-actin, some formins can additionally depolymerize, sever or bundle F-actin. Two mammalian formins, inverted formin 2 (INF2) and diaphanous 1 (DIA1, encoded by DIAPH1), exemplify this diversity. INF2 shows potent severing activity but elongates weakly whereas DIA1 has potent elongation activity but does not sever. Using cryo-electron microscopy (cryo-EM) we show five structural states of INF2 and two of DIA1 bound to the middle and barbed end of F-actin. INF2 and DIA1 bind differently to these sites, consistent with their distinct activities. The formin-homology 2 and Wiskott-Aldrich syndrome protein-homology 2 (FH2 and WH2, respectively) domains of INF2 are positioned to sever F-actin, whereas DIA1 appears unsuited for severing. These structures also show how profilin-actin is delivered to the fast-growing barbed end, and how this is followed by a transition of the incoming monomer into the F-actin conformation and the release of profilin. Combined, the seven structures presented here provide step-by-step visualization of the mechanisms of F-actin severing and elongation by formins.
History
DepositionMar 11, 2024-
Header (metadata) releaseMay 29, 2024-
Map releaseMay 29, 2024-
UpdateAug 21, 2024-
Current statusAug 21, 2024Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_44019.png

Downloads & links

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Map

FileDownload / File: emd_44019.map.gz / Format: CCP4 / Size: 216 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.08 Å/pix.
x 384 pix.
= 414.72 Å		1.08 Å/pix.
x 384 pix.
= 414.72 Å		1.08 Å/pix.
x 384 pix.
= 414.72 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.08 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.114
Minimum - Maximum-0.09956496 - 0.4545797
Average (Standard dev.)0.00008386066 (±0.016742889)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions384384384
Spacing384384384
CellA=B=C: 414.72003 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_44019_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Actin

EntireName: Actin
Components
  • Complex: Actin
    • Complex: INF2 Dimer
      • Protein or peptide: Inverted formin-2
      • Protein or peptide: Inverted formin-2
    • Complex: Actin Filament
      • Protein or peptide: Actin, alpha skeletal muscle
  • Ligand: ADENOSINE-5'-DIPHOSPHATE
  • Ligand: MAGNESIUM ION

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Supramolecule #1: Actin

SupramoleculeName: Actin / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#3
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 264 KDa

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Supramolecule #2: INF2 Dimer

SupramoleculeName: INF2 Dimer / type: complex / ID: 2 / Parent: 1 / Macromolecule list: #2-#3
Source (natural)Organism: Homo sapiens (human)

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Supramolecule #3: Actin Filament

SupramoleculeName: Actin Filament / type: complex / ID: 3 / Parent: 1 / Macromolecule list: #1
Source (natural)Organism: Oryctolagus cuniculus (rabbit)

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Macromolecule #1: Actin, alpha skeletal muscle

MacromoleculeName: Actin, alpha skeletal muscle / type: protein_or_peptide / ID: 1 / Number of copies: 7 / Enantiomer: LEVO
EC number: Hydrolases; Acting on acid anhydrides; Acting on acid anhydrides to facilitate cellular and subcellular movement
Source (natural)Organism: Oryctolagus cuniculus (rabbit)
Molecular weightTheoretical: 41.387227 KDa
Recombinant expressionOrganism: Oryctolagus cuniculus (rabbit)
SequenceString: TTALVCDNGS GLVKAGFAGD DAPRAVFPSI VGRPRHQGVM VGMGQKDSYV GDEAQSKRGI LTLKYPIE(HIC)G IITNWD DME KIWHHTFYNE LRVAPEEHPT LLTEAPLNPK ANREKMTQIM FETFNVPAMY VAIQAVLSLY ASGRTTGIVL DSGDGVT HN VPIYEGYALP ...String:
TTALVCDNGS GLVKAGFAGD DAPRAVFPSI VGRPRHQGVM VGMGQKDSYV GDEAQSKRGI LTLKYPIE(HIC)G IITNWD DME KIWHHTFYNE LRVAPEEHPT LLTEAPLNPK ANREKMTQIM FETFNVPAMY VAIQAVLSLY ASGRTTGIVL DSGDGVT HN VPIYEGYALP HAIMRLDLAG RDLTDYLMKI LTERGYSFVT TAEREIVRDI KEKLCYVALD FENEMATAAS SSSLEKSY E LPDGQVITIG NERFRCPETL FQPSFIGMES AGIHETTYNS IMKCDIDIRK DLYANNVMSG GTTMYPGIAD RMQKEITAL APSTMKIKII APPERKYSVW IGGSILASLS TFQQMWITKQ EYDEAGPSIV HRKCF

UniProtKB: Actin, alpha skeletal muscle

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Macromolecule #2: Inverted formin-2

MacromoleculeName: Inverted formin-2 / type: protein_or_peptide / ID: 2 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 6.870784 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString:
VPSHRRVNPP TLRMKKLNWQ KLPSNVAREH NSMWASLSSP DAEAVEPDFS SIERLFSFPA

UniProtKB: Inverted formin-2

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Macromolecule #3: Inverted formin-2

MacromoleculeName: Inverted formin-2 / type: protein_or_peptide / ID: 3 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 36.856391 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: KEPKEITFLD AKKSLNLNIF LKQFKCSNEE VAAMIRAGDT TKFDVEVLKQ LLKLLPEKHE IENLRAFTEE RAKLASADHF YLLLLAIPC YQLRIECMLL CEGAAAVLDM VRPKAQLVLA ACESLLTSRQ LPIFCQLILR IGNFLNYGSH TGDADGFKIS T LLKLTETK ...String:
KEPKEITFLD AKKSLNLNIF LKQFKCSNEE VAAMIRAGDT TKFDVEVLKQ LLKLLPEKHE IENLRAFTEE RAKLASADHF YLLLLAIPC YQLRIECMLL CEGAAAVLDM VRPKAQLVLA ACESLLTSRQ LPIFCQLILR IGNFLNYGSH TGDADGFKIS T LLKLTETK SQQNRVTLLH HVLEEAEKSH PDLLQLPRDL EQPSQAAGIN LEIIRSEASS NLKKLLETER KVSASVAEVQ EQ YTERLQA SISAFRALDE LFEAIEQKQR ELADYLCEDA QQLSLEDTFS TMKAFRDLFL RALKENKDRK EQAAKAERRK QQL AEE

UniProtKB: Inverted formin-2

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Macromolecule #4: ADENOSINE-5'-DIPHOSPHATE

MacromoleculeName: ADENOSINE-5'-DIPHOSPHATE / type: ligand / ID: 4 / Number of copies: 7 / Formula: ADP
Molecular weightTheoretical: 427.201 Da
Chemical component information

ChemComp-ADP:
ADENOSINE-5'-DIPHOSPHATE / ADP, energy-carrying molecule*YM

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Macromolecule #5: MAGNESIUM ION

MacromoleculeName: MAGNESIUM ION / type: ligand / ID: 5 / Number of copies: 7 / Formula: MG
Molecular weightTheoretical: 24.305 Da

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

Concentration1.45 mg/mL
BufferpH: 8
Component:
ConcentrationName
20.0 mMHEPES
50.0 mMNaCl
1.0 mMEDTA
1.0 mMDTT
0.05 %Thesit
GridModel: Quantifoil R1.2/1.3 / Material: COPPER / Mesh: 300 / Support film - Film thickness: 100 / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 120 sec.
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Number real images: 52360 / Average electron dose: 44.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: SPOT SCAN / Imaging mode: BRIGHT FIELD / Nominal defocus max: 2.5 µm / Nominal defocus min: 0.5 µm
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Startup modelType of model: NONE
Final reconstructionResolution.type: BY AUTHOR / Resolution: 3.82 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 4.0) / Number images used: 27770
Initial angle assignmentType: RANDOM ASSIGNMENT / Software - Name: cryoSPARC (ver. 4.0)
Final angle assignmentType: RANDOM ASSIGNMENT / Software - Name: cryoSPARC (ver. 4.0)
FSC plot (resolution estimation)

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